Besides anti-HLA antibodies, donor-specific antibodies against MICA and GSTT1 antigens could be responsible for the occurrence of antibody-mediated kidney graft rejection.

According to our results, patients with preformed or de novo anti-HLA-DSA showed the highest likelihood to suffer rejection episodes. Transplantation with preformed anti-HLA-DSA should be avoided, and an early detection of de novo HLA antibodies is important to treat patients before damage occurs in the graft.

Regulator of G-protein signalling (RGS)12 and -14 proteins possess the RGS domain, Ras-binding domains and the GoLoco motif. Emerging evidence suggests that these proteins are involved in several cellular functions in addition to stimulation of GTPase activity of G-protein alpha subunits. However, our understanding of the role of the two proteins in brain function remains marginal. Here, we have studied the expression pattern of RGS12 and RGS14 proteins in brain at regional, cellular and subcellular levels. Both proteins were expressed throughout the brain regions, including cortex, hippocampus, striatum, thalamus and substantia nigra. The most intense immunostaining for RGS12 was seen in cortex and that of RGS14 was found in striatum. In cortex, RGS12 and RGS14 proteins were associated with pyramidal and nonpyramidal cell types. Apical dendrites of pyramidal cells were also labelled. RGS12 was found in both nuclear and cytoplasmic compartments. In contrast to RGS12 protein, RGS14 was localized in astrocytes in addition to neurons. Pyramidal cells in the CA1 area showed labelling for both RGS proteins. The presence of RGS12 was predominantly nuclear in the striatum of rat brain; however, the labelling of this protein was non-nuclear in adult monkey brain. To our surprise, in 1-month-old monkey brain the immunostaining pattern of the same protein was changed to nuclear. Non-nuclear staining for RGS12 was also evident in thalamus of adult monkey brain; however, in 1-month-old monkey brain, it was seen into two different populations, one with nuclear and the other with cytoplasmic staining. Both RGS12 and RGS14 were exclusively localized at postsynaptic sites of excitatory synapses. Our results demonstrate a highly dynamic expression pattern of RGS12 and RGS14 proteins in the central nervous system, and support the view that these proteins may participate not only in G-protein receptor signalling pathways but also in other cellular activities.

The best combination of markers for the diagnostic screening of PNH would include evaluation of CD14 on monocytes and of CD16 on neutrophils, although further analysis of CD55 and CD59 expression may contain additional clinically useful information. Clear association between the genetic changes detected in the PIG-A gene in 5 of 13 cases analyzed, and the phenotypic profile of PNH cells has not been found. Additionally, an abnormally higher expression of several GPI-APs among normal residual cells from PNH patients in comparison to healthy donors was observed, suggesting that factors other than the PIG-A mutation could determine the phenotypic profile of PB cells in PNH.

Several gene delivery reagents were analyzed for their transfection efficiency. Genes studied belonged to the class of mammalian proteins termed regulators of G-protein signaling (RGS), ranged in size up to 2.2 Kb long and were transfected into the NG108-15, SH-SY5Y and CHO-K1 cell lines. Prior to transfection, genes were cloned into a nonviral vector pcDNA 6.2/EmGFP, so as to express a green fluorescent protein tag at the 3' end. Flow cytometry was used to analyze cell fluorescent activity and thereby transfection efficiency. Gene delivery reagents Lipofectamine 2000 and ExGen 500 produced more effective transfection in NG108-15 cells whereas Lipofectamine 2000, ExGen 500 and TurboFectin 8.0 were more effective in CHO-K1 cells. In both these cell lines, transfection efficiency reached 60-80%. In SH-SY5Y cells, TurboFectin 8.0 produced the best transfection result; however efficiency level was only 5%. Gene size had no effect on transfection efficiency. Unlike Lipofectamine 2000, cells transfected using ExGen 500 showed morphological deformation. Our results suggest that Lipofectamine 2000 is the most suitable transfection medium for gene delivery to NG108-15 and CHO-K1 cells.

Luminex bead-based assays are routinely used in the study of anti-human leukocyte antigen (HLA) donor-specific antibodies (DSA). Single antigen (SA) assays use beads coated with recombinant antigens whereas Luminex crossmatch (Xm-DSA) tests consist of beads coated with isolated donor-specific HLA molecules. The aim of this study was to compare these techniques used to detect DSA. A total of 24 sera recognizing different HLA class I (seven anti-HLA-A and seven anti-HLA-B) as well as class II (seven anti-HLA-DR and three anti-HLA DQ) specificities by complement dependent cytotoxicity were included in the study. These sera were used undiluted and in serial dilutions to perform both class I and II SA and Xm-DSA assays. In the case of Xm-DSA the same serum was checked with different lysates. A total of 42 lysates were used to perform a total of 61 crossmatches: 42 to detect anti-class I and 19 to detect anti-class II antibodies. The maximum positive dilution was higher for SA in 76% of the class I and in 90% of the class II crossmatches. Those cases with a higher sensitivity of the Xm-DSA could not be explained by a larger number of antigen targets.

Treatment of D2-receptor-expressing cells with specific drugs upregulates the receptor number at the cell surface independently of protein synthesis, leading to the concept of an intracellular receptor pool. However, how this pool is operating is still an enigma. Here, we report that a splice variant of the Gαi2 protein, protein sGαi2, plays a crucial role in the maintenance of this D2-receptor pool. Co-expression of sGi2 with D2 receptor reduced receptor localization to cell surface by one-third. This effect is associated with specific intracellular protein-protein interaction and the formation of a sGi2–D2-receptor complex. It has been suggested that the formation of this complex serves to prevent D2 receptors from reaching the cell membrane. Treatment of D2-receptor-expressing cells with agonists increased the number of cell surface D2 receptors and coincided with a reduction in these receptors from intracellular complexes, suggesting that agonist treatment released D2 receptors from the complex allowing them to localize to the cell membrane. Thus, in addition to elucidating how the intracellular pool of D2 receptor functions, our findings uncover a novel mechanism regulating the density of cell surface D2 receptors.

No abstract